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Making a new RIA/ELISA protocol - (Nov/30/2012 )

Hello everybody.

I been trying for a few weeks now to make up/finding a new protcol to replace our RIA protocol to measure insulin using 125-I (which works just fine)
but I've been asked to see if it's possible to melt together ELISA and RIA which we only have 1 Antibody, which we don't know too much about. (works against mice and rat insulin)
I have some plates made for immuno-assays and have been using an homemade PBS buffer both for dilution of my antibodies and samples. Washing with PBS buffer w. 1 v/v% BSA + 0,05v/v% TWEEN 20 pH 7.4.

I've tried with different times like; 2x3 (2h coat - 2h block - 2h sample/tracer), 4x2+24, 4x3, 24x3. Higher antibody concentration, Keeping them at 4, room and 37 degrees C.

We only have that one antibody and need to make it work only with that.

I'm about run out of idea and starting to doubt it can be done with only; coating(Antibody) -> Block(PBS w. BSA only) -> Samples+Tracer -> add a special liquid and then read on gammacounter.

Anybody got some ideas?



Not sure exactly what you are having issues with and I think that's why this post has not received any comments. Can you share some results and specifically state what issues/problems are at hand?